肉認証にペプチド応用のMRM質量分析法を用いて混合物中の種の決意と定量
Summary
We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.
Abstract
We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of ‘corresponding proteins’ in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are ‘corresponding peptides’. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.
Introduction
The European horse meat scandal of 2013, in which undeclared horse meat was found in a number of supermarket beef products1, highlights the need for testing methods capable of detecting and measuring food fraud in meat. Several technologies have been explored, especially enzyme-linked immunosorbent assay (ELISA) and DNA-based methods2. An alternative route, based on mass spectrometry, targets species-specific peptides which in turn arise from species-specific proteins. Here we outline one such peptide-based approach that offers both identification and relative quantitation of the adulterant species in a meat mixture3.
The protocol is framed in the context of red meats and the desire to determine the presence of one in another at the level of 1% by weight, the level considered by some to represent fraudulent food adulteration as opposed to contamination4. The method relies in the first instance on identifying a protein which is nominally ‘the same’ in all target meats. Myoglobin, the protein responsible for the red color of meat, is a good candidate since it is abundant, relatively heat tolerant and water soluble, and has been used for species determination of meat previously5,6. The myoglobins for beef (Bos Taurus), pork (Sus scrofa), horse (Equus caballus) and lamb (Ovis aries)3, for instance, are nominally the same, as required, but their sequences are not identical. Such groups of ‘similar but different’ proteins, like these four myoglobins, can conveniently be described as ‘corresponding proteins’. The sequence differences in these four myoglobins are species-specific: for example, the full myoglobin proteins for beef and horse, P02192 and P68082 respectively, each comprise 154 amino acids with 18 sequence differences between the two. Subject to proteolysis using trypsin these proteins produce two sets of peptides, some of which are identical, and some which show one or more species-specific amino acid differences: corresponding proteins therefore give rise to corresponding peptides.
The CPCP approach, therefore, seeks first to identify proteins from two or more species where these proteins exhibit limited species-specific sequence variants. These are corresponding proteins. Following proteolysis, corresponding proteins give rise to peptides, some of which likewise display species-specific sequence variants inherited from the parent protein. These are corresponding peptides. The CPCP approach can be used to compare levels of two corresponding proteins in a mixed species sample by monitoring the levels of corresponding peptides.
The natural technology for the detection of known peptides is multiple reaction monitoring mass spectrometry, or MRM-MS7. Species-specific peptides yield precursor ions, which along with their mass spectrometry fragment ions, are easily itemized in advance by software tools. These lists are then used to instruct the mass spectrometer to record only specific precursor plus fragment ion pairs, called transitions. A particular target peptide is therefore identified not only by its retention time in the chromatography preceding the mass spectrometer, but also by a set of transitions sharing a common precursor ion. This is a highly selective means of detecting known peptides that makes efficient use of the mass spectrometer resource.
Other authors have used mass spectrometry to test for meat adulteration via peptide markers but from disparate proteins8-14. Using the corresponding proteins, corresponding peptides (CPCP) scheme, however, means experimental conditions can be optimized, aiding identification of the species in the mixture from known species-specific transitions. In addition, corresponding proteins and peptides will generally behave similarly in the extraction, proteolysis and detection stages. Since transition peak areas are quantitative and reproducible, ratios of peak areas arising from pairs of corresponding peptides from different species provide a direct estimate of the relative quantities of two meats in a mixture. In contrast, more traditional quantitation routes exploit calibrations based on reference materials to establish absolute quantitation14,15.
Though the protocol is outlined in the context of myoglobin and meat, proteins other than myoglobin could be used for identification and relative quantitation via the CPCP strategy in meat mixtures, though potentially with modifications to the protocol. In addition the strategy is also applicable to binary mixtures of other species sharing one or more corresponding proteins.
The starting point for the protocol is purified ‘reference’ myoglobin, which for some species can be purchased but which for others must be prepared by conventional size-exclusion chromatography. The procedure for preparing reference myoglobin is not included in the protocol, but is described elsewhere3. Software tools16 are used to list candidate peptides and transitions arising from myoglobins of interest. Each reference myoglobin is subjected to proteolysis and the resultant peptides analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to discover which of the candidate precursor ions and transitions are most useful, and to determine the matching peptide retention times. The outcome of this stage is a revised list of target peptides with their transitions, suitable for species determination, and a list of CPCP pairs, suitable for relative quantitation. To test real meats, sample extractions are prepared then subjected to proteolysis to generate peptides both from myoglobin and other extraneous proteins. The myoglobin-based peptides are then monitored by LC-ESI-MS/MS based on their listed transitions. The species present in a mixture are identified by the transition peaks associated with marker peptides. Estimates of the relative amounts of two meats in a binary mixture are calculated using ratios of transition peak areas. A set of test mixtures of pairs of meats will allow the ratio of peak areas for a given pair of transitions to be checked and calibrated against actual mixtures.
Protocol
Representative Results
Discussion
好適な標的タンパク質の選択が重要です。優れた標的タンパク質は、目的の種の対応する形態は、十分な種依存性配列変異、種特異性を有し、生物内でアクセス可能な量で存在する必要があります。処理を受けた混合物（例えば、熱処理）を評価するため、その処理に比較的免疫配列を有するタンパク質であることが望ましいです。ミオグロビンは、調理された赤身肉などの赤肉、のための良い候補であるが、唯一の可能性ではありません。標的タンパク質が決定されると、プロトコルの最も重要な部分は、タンパク質のタンパク質分解です。ミオグロビンは異なるタンパク質がよく、代替タンパク質分解プロトコルを要求することができます。
記載されているようにプロトコルは、基準精製タンパク質に基づいてセグメントを含みます。これは、保持時間ウィンドウと、適切な前駆体およびフラグメントイオンを発見することを目指しています。このセグメントは非常に有用であるが、必須ではありません。
<p関心の2種からの対応するペプチドのペアがあっても実験することなく記載されていることができますが、クラス= "jove_contentは">、それは時々、配列の差が消化プロファイルに劇的な影響を有している場合です。例えば、ペプチドペアVLGFHG（牛肉）とELGFQG（馬）（ 図2の1未満の勾配としてマニフェスト）異常 な定量結果を与えます。後者のペプチドは、混合物中の馬のレベルの過少見積もりを引き起こし、比較的抑制KE切断から生じるためです。異なるアミノ酸で始まる対応するペプチドは、したがって、最高の回避されます。多くの場合、対応する二つのペプチドのフラグメントは、同一のアミノ酸配列を有し、行儀されているが、これは常にそうではなく、方法の開発中にチェックする必要があります。種の同定は、相対定量よりも、これらの問題に非常に敏感ではありません。プロトコルは、4つの赤肉のために実証されています秒3。あまりにも多くのマーカーペプチドが共溶出場合、遷移ピーク形状の品質を効果的に滞留時間を減少させ、最終的に相対的な定量推定値を分解、劣化するかもしれないが、追加の肉種が、含まれ得ます。すでに利用可能な改善された機器は、これを改善します。関連する問題ではない、すべての肉は、異なるミオグロビンを有することです。例えば、馬、ロバとシマウマミオグロビンは同一であり、したがって、厳密方法を言えば牛肉に馬やロバやシマウマを検出することができるだけです。いくつかのケースでは、ミオグロビンが同一ではないにもかかわらず、いくつかの重要なペプチドは、することができます。例えば、いくつかのラムミオグロビン由来のマーカーペプチドはまた、ヤギに表示されます。
このおよび他のタンパク質ベースの定量法が直面している合併症は、タンパク質またはペプチドのレベルは、混合物中の肉のレベルに自明と同一視することがある場合、タンパク質レベルは、全ての種にわたって一定であると仮定しなければならないことです。ミオグロビンと4赤メートルのためにこれは普遍的真実ではありません食べます。一般的にレベルは豚肉は4の最低レベルを示すと、依存種です。また、ミオグロビンレベルは肉カットと動物の年齢に応じて変化します。遷移のピーク面積の比が、ミオグロビンの比率に確実にマッピングがので、実際の肉の割合へのマッピングは、混合物中の肉の可能性ソースに関する仮定の推定図です。
この作業で概説したアプローチは、他の公開の貢献から、いくつかの方法で異なっています。より典型的な経路は、異なる種のマーカーが互いに8-12,14,19とは特定の関係を有していない、その場合には、様々な異種の種依存性マーカーペプチドを同定するプロテオミクスの方法を使用することです。これとは対照的に、我々は3バリアント 、種依存性シーケンスまで関心のあるすべての種に共通するタンパク質を選択しました。別に私たちの相対定量戦略の中心であることから、これはそのサンプル利点を有しています調製ストラテジーを最適化することができます。さらに、このような対応するタンパク質は、抽出、またはそのような調理または缶詰などのサンプルの商業的処理、例えば、同様に挙動することが予想されるかもしれません。種の同定は、その後、通常、典型的には、1つまたは2配列の差異を有する密接に関連するペプチドの検出によりCPCPアプローチ種の識別が進む中で、一方、異種のマーカーペプチドの検出を介して進行します。最後に、他に1種の重量％を推定するタンパク質の定量は、従来別々に既知の標準7,14,15に基づいて、各タンパク質の絶対定量を介して進行することがあります。しかし、較正方法を必要としないCPCP法を用いて。代わりに、相対的なレベルは、完全に絶対測定ステージをバイパスし、両種からの対応する二つのペプチドの信号強度を比較することによって推定されます。究極の目標は、あの、1つの種の重量％であることからTHER、相対定量、その後CPCPは両方のより直接的かつ2つの絶対定量測定値を比較するよりも簡単です。これらの機能は、食品の不正検出の分野における急速な監視ツールとしての技術が有用なもの、洗練されたプロトコルを用いて、約2時間であることが予想される短い実験時間、に変換されます。
Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge financial support from Institute of Food research BBSRC Core Strategic Grant funds, BBSRC Project BB/J004545/1.
Materials
| Uniprot database | www.uniprot.org | Freely accessible database of protein sequences | |
| Skyline software | www.skyline.gs.washington.edu | Free software to download that enables the creation of targeted methods for proteomic studies, peptide and fragment prediction | |
| Ammonium bicarbonate | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | O9830 | |
| Methanol, HPLC grade | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 10674922 | |
| Acetonitrile, HPLC grade | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 10010010 | |
| Urea | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | U5378 | |
| Trypsin(from bovine pancreas treated with TPCK) | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | T1426 | |
| Formic acid | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | F0507 | |
| Coomassie Plus Protein Assay Reagent | Thermo Fisher Scientific www.thermofisher.com | 1856210 | |
| Protein standard | Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com | P0914 | |
| Ultra Turrax homogeniser T25 | Fisher Scientific, Loughoborough, UK www. fisher.co.uk | 13190693 | |
| Edmund and Buhler KS10 lab shaker | |||
| Heraeus Fresco 17 Centrifuge | Thermo Fisher Scientific www.thermoscientific.com | 75002420 | |
| Vacuum centrifuge RC 1022 | Jouan | ||
| Plate Reader | |||
| Strata-X 33u polymeric reversed-phase cartridges 60 mg/3 ml tubes | Phenomenex, Macclesfield, UK | 8B-S100-UBJ | |
| 4000 QTrap triple-quadrupole mass spectrometer | AB Sciex, Warrington, UK www.sciex.com | ||
| 1200 rapid resolution LC system | Agilent, Stockport, UK | ||
| XB C18 reversed-phase capillary column (100 x 2.1mm, 2.6µ particle size) | Phenomenex, Macclesfield, UK www.phenomenex.com | ||
| Analyst 1.6.2 software | AB Sciex, Warrington, UK www.sciex.com | QTrap data acquisition and analysis, including peak area integration | |
| Autosampler vials |
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